Fluidic device for aliquoting and combinatorial mixing of liquids

ABSTRACT

The present invention relates to fluidic devices, especially microfluidic devices, for aliquoting and pairwise combinatorial mixing of a first set of liquids with a second set of liquids. The device architecture is designed to move liquids in two separate phases, a first phase where the liquids are exposed to a first directional force field to move the liquids in a first direction, from a reservoir to aliquot chambers, and a second phase where the liquids are exposed to a second directional force field to move the liquids in a second direction, from the aliquot chambers to the mixing chambers. The first and second directional force fields that the device is exposed to may be achieved using a single directional force field (i.e. a rotor driven centrifugal force field) and by re-orienting the position of the device with respect to the centrifugal forces between the first and second phases of operation. The device architecture comprises reservoirs for each of the first fluids and reservoirs for each of the second fluids. Each reservoir is fluidically connected to aliquoting chambers, either arranged in parallel or in series, for providing aliquots of the fluid which may be metered. The conduits providing fluid communication between the reservoirs and aliquoting chambers are arranged in a first direction. A series of mixing chambers is also provided, and each mixing chamber is fluidically connected to one aliquot chamber for a first liquid and one aliquoting chamber for a second liquid. The conduits providing fluid communication between the aliquoting chambers and mixing chambers are arranged in a second direction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/EP2017/080708 filed Nov. 28, 2017,which designates the U.S. and claims benefit under 35 U.S.C. § 119(a) ofGB Provisional Application 1620320.0 filed Nov. 30, 2016, the contentsof which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a fluidic device for aliquoting andcombinatorial mixing of a plurality of first liquid samples with aplurality of second liquid samples. Also contemplated are methods ofmetering and combinatorial mixing of first and second liquid samples.

BACKGROUND TO THE INVENTION

On a centrifugal fluidic platform^(1, 2), a centrifugal force is inducedby rotating a rotor-based fluidic chip which may be a disc-shaped withsimilar geometry as common CD or DVD optical drives. These so-called“Lab-on-a-Disc” (LoaD) platforms have been engineered towards a varietyof applications such as biomedical diagnostics³, bioprocess monitoring⁴and environmental screening⁵⁻⁷. By virtue of their ease-of-use of samplehandling absence for pressurised fittings or external pumps, these LoaDsystems are particularly useful for near-patient/point-of-care/point ofuse applications.

Advanced flow control is key in centrifugal microfluidics as all liquidvolumes residing on the spinning disc are subject to the centrifugalfield. The omnipresence of this field drives all on-board liquidssimultaneously outwards, thus often requiring on-disc valving schemesfor the temporal control of liquid handling sequences. Such valving canbe actuated by changing the centrifugal force acting on the liquid, e.g.by changing the spin rate. Several methods such as surface tension, caninclude capillary burst valves⁸⁻¹², dissolvable film (DF)valves^(13-16,) burstable foils¹⁷, elastomeric membranes¹⁸ and dead-endpneumatic chambers¹⁹, siphons^(4, 20, 21) and pneumatically enhancedsiphons²²⁻²⁵ have been developed which are primarily based upon theinterplay between centrifugally induced hydrostatic pressure and otherforces.

An alternative approach is to use a peripheral module to interact withthe disc, either while the disc stationary or under rotation. Knownmethods of such instrument-based, non-centrifugal actuation includeexternal pressure sources^(5, 26,) thermal energy or radiation, e.g. toinduce phase-changes²⁷⁻³⁰ in wax plugs or ablate sealing films. Flowcontrol has also been implemented by varying the orientation of a chipattached to the rotor within the centrifugal field³¹⁻³⁴.

SUMMARY OF THE INVENTION

The present invention relates to fluidic devices, especiallymicrofluidic devices, for aliquoting (and in one embodiment, metering)and pairwise combinatorial mixing of a first set of liquids with asecond set of liquids. The device architecture is designed to moveliquids in two separate phases, a first phase where the liquids areexposed to a first directional force field to move the liquids in afirst direction, from a reservoir to aliquot (or metering) chambers, anda second phase where the liquids are exposed to a second directionalforce field to move the liquids in a second direction, from the aliquotchambers to the mixing chambers. The first and second directional forcefields that the device is exposed to may be achieved using a singledirectional force field (i.e. a rotor driven centrifugal force field)and by re-orienting the position of the device with respect to thecentrifugal forces between the first and second phases of operation. Thedevice architecture comprises reservoirs for each of the first fluidsand reservoirs for each of the second fluids. Each reservoir isfluidically connected to aliquot chambers, either arranged in parallelor in series, for providing aliquots of the fluid. The conduitsproviding fluid communication between the reservoirs and aliquotchambers are arranged in a first general direction. A series of mixingchambers is also provided, and each mixing chamber is fluidicallyconnected to at least one aliquot chamber for a first liquid and atleast one aliquot chamber for a second liquid. The conduits providingfluid communication between the aliquot chambers and mixing chambers arearranged in a general second direction. In one embodiment, the aliquotchambers are configured to provide metered aliquots of the liquids.

As an example, a device for providing metering and combinatorial mixingof three (M) first liquids (i.e. three liquid samples) with three (N)reagents is described. The device comprises six (M+N) reservoirs, onefor each of the liquid samples and one for each of the reagents. Eachreservoir is fluidically connected to three metering chambers byconduits in a first direction, thus there are 18 metering chambers. M*N(i.e. 9) mixing chambers are provided, and each mixing chamber isfluidically connected to a different pair of metering chambers.Application of a centrifugal field that is aligned with the firstdirection causes the metering of each liquid sample and reagent intothree aliquots of each. Re-orientation of the device such that thecentrifugal field is aligned with the second direction causes thealiquots of sample and reagent to move into the mixing chambers. Anexample is illustrated with reference to FIG. 4.

To provide multiple aliquots of each liquid sample, and combinatorialmixing of the aliquots, in two phases using a first directional forcefield to move liquid in metering operation and a second directionalforce field to move liquid in a mixing operation, generally requiresthat either the metering conduits, or mixing conduits, cross paths. Thisis facilitated by providing the conduits, or at least parts of theconduits, in different planes of the device. For example, with referenceto FIG. 4, the four liquid samples are metered into the sixteen meteringchambers by means of conduits located in a first plane (i.e. layer) ofthe device, and the four reagents are metered into sixteen meteringchambers by means of conduits located in a second plane (i.e. layer) ofthe device. The mixing region of metering/mixing chamber extends acrossthe layers of the device. This provides Y-axis dimensional bridging ofthe conduits, allowing multiple samples and reagents to becombinatorially mixed.

According to a first aspect, the invention provides a fluidic device foraliquoting and combinatorial mixing of a number M of first liquidsamples with a number N of second liquid samples, the microfluidicdevice comprising:

first reservoirs for each of the M first liquid samples and secondreservoirs for each of the N second liquid samples;

N first aliquot chambers fluidically connected to each of firstreservoirs by first microfluidic conduits having a first orientationrelative to the device;

M second aliquot chambers fluidically connected to each of the secondreservoirs by second microfluidic conduits having a first orientationrelative to the device; at least M times N mixing chambers, in whicheach mixing chamber is fluidically connected to at least one of the Nfirst aliquot chambers and at least one of the M second aliquot chambersby third microfluidic conduits having a second orientation relative tothe device.

Typically, the first and second microfluidic conduits are configuredsuch that application of a first unidirectional force field effectsliquid flow in the first orientation to generate aliquots of the M firstliquid samples in the N first aliquot chambers and N second liquidsamples in the M first aliquot chambers.

Typically, the third microfluidic conduits are configured such thatapplication of a second unidirectional force field effects liquid flowin the second orientation to combinatorially combine aliquots of the Mfirst liquid samples with aliquots of the N second liquid samples.Generally, at least some of the conduits are spatially separated acrossthe Y-axis of the device.

In another aspect, the invention provides a planar layered microfluidicdevice for aliquoting and mixing of a plurality of first liquid sampleswith a plurality of second liquid samples, the planar layeredmicrofluidic device comprising:

first reservoirs for each of the first liquid samples and secondreservoirs for each of the second liquid sample;

first aliquot chambers fluidically connected to each of first reservoirsby first microfluidic conduits having a first orientation relative tothe device;

second aliquot chambers fluidically connected to each of the secondreservoirs by second microfluidic conduits having a first orientationrelative to the device;

a plurality of mixing chambers, in which at least some of the mixingchamber are fluidically connected to one of the first aliquot chambersand one of the second aliquot chambers by third microfluidic conduitshaving a second orientation relative to the device

whereby the first and second microfluidic conduits are configured suchthat application of a first directional force field effects liquid flowin the first and second conduits but not in the third conduits, andwhereby the third microfluidic conduits are configured such thatapplication of a second directional force field effects liquid flow inthe third microfluidic conduits but not in the first or second conduits,wherein at least some of the conduits are spatially separated across theY-axis of the device.

In a further aspect, the invention provides a fluidic device foraliquoting and combinatorial mixing of a number M of first liquidsamples with a number N of second liquid samples, the microfluidicdevice comprising:

first reservoirs for each of the M first liquid samples and secondreservoirs for each of the N second liquid samples;

N first aliquot chambers fluidically connected to each of firstreservoirs by first microfluidic conduits having a first orientationrelative to the device;

M second aliquot chambers fluidically connected to each of the secondreservoirs by second microfluidic conduits having a first orientationrelative to the device;

at least M times N mixing chambers, in which each mixing chamber isfluidically connected to at least one of the N first aliquot chambersand at least one of the M second aliquot chambers by third microfluidicconduits having a second orientation relative to the device,

wherein, the first and second microfluidic conduits are configured suchthat application of a first unidirectional force field effects liquidflow in the first orientation to generate aliquots of the M first liquidsamples in the N first aliquot chambers and N second liquid samples inthe M first aliquot chambers.

wherein, the third microfluidic conduits are configured such thatapplication of a second unidirectional force field effects liquid flowin the second orientation to combinatorially combine aliquots of the Mfirst liquid samples with aliquots of the N second liquid samples.wherein at least one of the first reservoirs is fluidically connected tothe N first metering chamber in series, and wherein at least some of theconduits are optionally spatially separated across the Y-axis of thedevice.

The number M may be 1 and preferably is at least 2 or 3. The number N istypically at least 2 or 3. In one embodiment, M is at least 3 and N isat least 3. In one embodiment, M is at least 4 and N is at least 4. Mand N may be different numbers, for example, the device of the inventionmay be configured for combinatorial mixing of 3 samples (first liquidsamples) with 4 reagents (second liquid samples). The term liquid samplemay refer to any type of liquid, for example a biological fluid, apharmaceutical sample, a chemical, a reagent or a buffer. The differentliquids may also contain the same type of substance, but at a variety ofconcentrations.

In one embodiment, the aliquot chambers are metering chambers configuredto provide metered aliquots of liquid. Methods of providing meteredaliquots of liquids are described herein, although it will beappreciated that the aliquot chambers of the invention may or may notprovide for metering of liquids. Thus, while the descriptionpredominantly refers to metering chambers, it will be appreciated thatthe term may be replaced in parts or throughout with aliquot chambers.

In one embodiment, at least one of the first reservoirs is fluidicallyconnected to the N first metering chambers in parallel. This means thatthe metering chambers are directly fluidically connected to thereservoir. In one embodiment, at least one, and ideally all, of themetering chambers are fluidically connected to a fluid sink, typicallybe means of an overflow mechanism.

In another embodiment, at least one of the first reservoirs isfluidically connected to the N first metering chamber in series (i.e. ametering chamber A is fluidically connected to the reservoir, meteringchamber B is fluidically connected to metering chamber A, meteringchamber C is fluidically connected to metering chamber B, etc.). In oneembodiment, the metering chambers are fluidically connected by means ofan overflow mechanism which serves to meter the liquid in the meteringchamber. Typically, the overflow mechanism is provided by an outlet inthe metering chamber which defines the volume of liquid retained in themetering chamber. This is illustrated in FIG. 5C. In one embodiment, thefinal metering chamber in the series has an overflow fluidicallyconnected to a waste chamber.

In one embodiment, at least one of the mixing chambers and fluidicallyconnected first and second metering chambers are provided by a singlechamber having aliquot or metering regions and a mixing region disposedsuch that application of the second directional force field effectsliquid flow from the aliquot or metering regions to the mixing region.Examples of single metering/mixing chambers are provided in FIGS. 5, 12and 13.

In one embodiment, the aliquot or metering regions and mixing region aredisposed on opposite ends of the single chamber.

In one embodiment, the single chamber has a generally arch-shape,wherein the feet of the arch-shaped chamber comprise the aliquot ormetering regions and the apex of the arch-shaped chamber comprises themixing chamber.

In one embodiment, at least some of the conduits are disposed on a firstplane of the device, and at least some of the conduits are disposed on asecond plane of the device. This allows the conduits cross each otherduring the aliquoting/metering step providing Y-axis dimensionalbridging, as illustrated in FIG. 4.

In one embodiment, the first conduits (fluidically connecting the firstreservoirs with the first metering chambers/regions) are disposed on afirst plane of the device, and the second conduits (fluidicallyconnecting the second reservoirs with the second meteringchambers/regions) are disposed on a second plane of the device. In oneembodiment, the mixing chamber spans the first and second planes of thedevice.

In one embodiment, at least some of the third conduits are disposed on afirst plane of the device, and at least some of the third conduits aredisposed on a second plane of the device. This embodiment is usefulwhere the first and second conduits are disposed on the same plane ofthe device, as it provides for Y-axis dimensional bridging.

In one embodiment, one or more of the conduits has at least one part inone plane of the device and at least a second part in a second plane ofthe device, and at least one orthogonal component providing fluidicconnection between the at least first and second parts.

In another embodiment, at least one of the conduits comprises a distaland proximal part in one plane of the device, and an intermediate partin a second plane of the device, and first and second orthogonalcomponents providing fluidic connection between the distal and proximal,and intermediate, parts of the conduit.

In one embodiment, at least one of the conduits comprises an orthogonalcomponent providing fluidic connection between the conduit and a fluidicchamber (for example a reservoir, aliquoting or metering chamber, ormixing chamber).

In one embodiment, one or more of the first metering chambers aredisposed in a first plane of the device and one or more of the secondmetering chambers are disposed in a second plane of the device.

In one embodiment, one or more of the mixing chambers span the first andsecond planes of the device.

In one embodiment, at least one of the mixing chambers comprises a trapstructure configured to prevent liquid in the mixing chamber returningto the associated metering chambers upon application of the firstdirectional force field. An example of a trap structure is provided inFIG. 16.

In one embodiment, the device comprises a liquid collection chamberfluidically connected the trap structures of the mixing chambers bycollection conduits, wherein the collection conduits are configured toforce the liquid in the mixing chambers to the collection chamber uponapplication of a directional force field, for example the firstdirectional force field.

In one embodiment, the device comprises a third reservoir for a thirdliquid sample, and a conduit configured to deliver an aliquot of thethird liquid sample to one or more of the mixing chambers. In oneembodiment, the conduit is provided in a third plane of the device. Inone embodiment, a pumping mechanism is provided to pump the third liquidsample from the reservoir to the metering/mixing chambers. An example isshown in FIG. 9.

In one embodiment, the first and second conduits are configured to havea higher fluidic resistance compared with the third conduits.Advantageously, this has the effect of throttling liquid flow throughthe device, and prevents liquid overflowing the metering chambers intothe third conduits during application of the first directional forcefield.

In one embodiment, the first and second conduits are disposed generallylaterally with respect to the chip and the third conduits are disposedgenerally longitudinally with respect to the chip. In one embodiment, inwhich the directional force field is a rotor-driven centrifugal field,the device is positioned during the metering phase with the thirdconduits extending generally radially inwards (thus against thedirection of the centrifugal force). This prevent movement of out of themetering chambers during the metering operation.

In one embodiment, the metering chambers are arrayed in a diamondconfiguration (see FIG. 20.1 for example). In one embodiment, thereservoirs and combined metering/mixing chambers are arrayed in adiamond configuration (See FIGS. 20.2 and 20.3). In one embodiment, thereservoirs are disposed on adjacent sides of the diamond array. Theseembodiments provide a diagonal orientation of first and second conduits,which has been found to meter and transfer fluid as effectively asvertical conduits, and avoids difficulties associated with horizontalconduits. Three different diamond arrays are illustrated in FIG. 20.

In one embodiment, the device is configured to combinatorially mix atleast one liquid sample with a second liquid sample in unequal mixingratios. For example, two volumes of a first liquid sample may be mixedwith one volume of a second liquid sample

In one embodiment, the device is formed from a microtitre plate (i.e.for example, a common off the shelf 96 well plate) and an attachment forthe plate (for example a closing lid) comprising the reservoirs,aliquot/metering chambers, and conduits, wherein the wells of the plateare the mixing chambers.

In one embodiment, the attachment comprises a first region comprisingreservoirs configured to receive the first and second liquid samples,and a second region configured to attach to a top of the microtiterplate to close some or all of the wells of the plate. In one embodiment,an underside of the second region comprises a plurality of spoutsconfigured to project into wells of the plate when the device isassembled. In one embodiment, the attachment comprises thealiquot/metering chambers and the first and second conduits providingfluidic connection between the reservoirs and aliquot/metering chambers.In one embodiment, the third conduits provide fluidic connection betweenthe aliquot/metering chamber and the spouts.

In one embodiment, the attachment comprises a third region comprising aplurality of waste chambers configured to receive excess liquids fromthe aliquot/metering chambers. In one embodiment, the first and thirdregions are disposed at each end of the attachment, and the secondregion is disposed between the first and third regions.

In use, the device is assembled and the reservoirs are loaded withliquids, before the device is attached to a rotor, for example a flyingbucket rotor centrifuge. The device is offset (i.e. 45 degrees) so thata component of the centrifugal force acts laterally. The device iscentrifuged in this position, forcing liquids from the reservoirs intothe aliquot/metering chambers. The rotor is then stopped and the deviceis re-positioned, and a second centrifuge step is carried out whichforces liquid from the aliquot/metering chambers into the wells of themicrotiter plate through the spouts, where aliquots of the liquids mix.

In this embodiment, the first and second directional force fields can beachieved with a common laboratory flying bucket-type centrifuge.

In one embodiment, the device is a planar device (i.e. chip) having aplurality layers (planes).

In one embodiment, the fluidic device is a microfluidic device.

The invention also provides a method of aliquoting (or metering) andcombinatorial mixing of a number M of first liquid samples with a numberN of second liquid samples, which method employs a fluidic deviceaccording to the invention, the method comprising the steps of:

exposing the fluidic device to a first direction force field whereby analiquot of the M first liquid samples is forced into the N firstaliquoting/metering chambers and an aliquot of the N first liquidsamples is forced into the M second aliquoting/metering chambers;

exposing the fluidic device to a second directional force field wherebythe aliquots of the first and second liquid samples are forced into themixing chambers in a combinatorial manner.

In one embodiment, the first and second directional force fields areeach, independently, selected from a centrifugal force field and agravitational force field or a combination of both.

In one embodiment, the first and second directional force fields aregenerated by changing the orientation of the device in a commondirectional force field. In one embodiment, reorientation is implementedmanually. In one embodiment, reorientation is implemented by changing acharacteristic of the directional force field, for example changing therotational speed or acceleration when the force field is centrifugal. Inone embodiment, reorientation of the device is performed using a passivemechanical spring- or magnet-based mechanism, or by an active mechanism(i.e. a mechatronic mechanism).

In one embodiment, the common directional force field is selected from acentrifugal force field and gravity, wherein the method includes thesteps of disposing the device in a first orientation in the commondirectional force field to generate the first directional force field,and then disposing the device in a second orientation in the commondirectional force field to generate the second directional force field.

In one embodiment, the first liquid samples are biological samples andthe second liquid samples are reagents.

Other aspects and preferred embodiments of the invention are defined anddescribed in the other claims set out below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates how different centrifugal forces can be applied tothe chip by reorienting the chip with respect to the centrifugal force.In this case the chip is rotated through 45 degrees.

FIG. 2 illustrates how different centrifugal forces can be applied tothe chip by reorienting the chip with respect to the centrifugal force.In this case the chip is rotated about an axis that runs perpendicularto the direction of the centrifugal force.

FIG. 3 illustrates a mechanism for re-orienting the chip based onvarying the centrifugal force. Initially the chip is held in location bya magnetically based latch. Following the first centrifugation,increasing the centrifugal force overcomes the magnetic force and thechip flips about its axis. This re-directs the centrifugal forcerelative to the chip orientation. Note this could also be implementedthrough an external input to open the latch, by incorporating a springin the axis or by other means.

FIG. 4 illustrates a chip according to the invention that is configuredfor pairwise combinatorial mixing of four first liquids with four secondliquids. The chip is shown in an expanded view illustrating thedifferent layers of the device and the separation of the first andsecond conduits into different layers of the device (Y-axis dimensionalbridging). The layers of FIGS. 4A and 4G are front and rear coverlayers, respectively, the front cover having apertures for filling thereservoirs. The layer of FIG. 4B contains the first conduits providingfluidic connection between the first reservoirs and mixing chambers.This figures also illustrates how each reservoir is fluidicallyconnected to four metering/mixing chambers in series by means of anoverflow mechanism. FIGS. 4C to 4E illustrates how the circular mixingregion of the metering/mixing chamber spans the three layers of FIGS. 4Cto 4E, and one metering chamber is disposed in the layer of FIG. 4C andone is disposed in the later of FIG. 4E, which ensures that the meteredaliquots in the metering chambers are kept separate until a seconddirectional force is applied which forced the respective aliquotstowards the mixing region where they combine. The layer of FIG. 4Fcontains the second conduits providing fluidic connection between thesecond reservoirs and metering/mixing chambers.

FIG. 5 illustrates a combined metering/mixing chamber forming part ofthe microfluidic chip of FIG. 4, in which the first metering region isdisposed in a first plane of the device, the second metering region isdisposed in a second plane of the device, and the mixing region spansthe first and second planes.

FIG. 6 illustrates a combined metering/mixing chamber similar to theembodiment of FIG. 5 but having four metering pockets.

FIG. 7 illustrates a chip according to the invention that is similar tothe chip of FIG. 4 but is configured for pairwise combinatorial mixingof three first liquids with three second liquids and includesarch-shaped metering/mixing chambers (illustrated in more detail in FIG.8). The chip is mounted on an arm: FIG. 5A illustrates the liquidsamples loaded into the reservoirs, FIG. 5B illustrates the metering ofthe liquid samples into aliquots in the metering chambers with the chipin the first orientation with respect to the rotor, whereby thecentrifugal force causes the liquid samples to move in a first directiontowards the metering chambers, and FIG. 5C shows the mixing step wherethe chip is reoriented into a second orientation (rotated through 180degrees) whereby the centrifugal force causes the liquid samples to movein a second direction from the metering chambers to the mixing chamber.In this embodiment, each of the mixing chambers, and associated meteringchamber, are provided by a single combined metering/mixing chamberhaving an inverted V-shape and metering pockets and an opposed mixingpocket. successful mixing occurred. In some cases, due to manufacturingflaws, less liquid than desired was metered and a dominant colour can beseen (red or green).

FIG. 8 illustrates in more detail the combined metering/mixing chamberforming part of the chip illustrated in FIG. 7. FIGS. 8A to 8Dillustrates liquid flow in response to the first directional forcefiled, where an aliquot of a first liquid sample and second liquidsample is forced into the metering pockets of the metering/mixingchamber where the overflow outlets allow excess fluid to leave themetering pockets (FIG. 8C) leaving a metered aliquot of the respectivefluids (FIG. 8D). The device is then re-oriented with respect to thedirection of the centrifugal field whereby the centrifugal forces(second directional force field) forces the liquids in the meteringpockets into the mixing pocket (FIGS. 8E and 8F).

FIG. 9 illustrates an embodiment of the invention having separatemetering and mixing chambers. In this case the mixing chambers areextended so that, during the first centrifugation, the geometricbarriers extend radially inwards of the loading chambers. In theconfirmation shown here the design will array 1 liquid with 2 liquids.

FIG. 10 illustrates one embodiment of a microfluidic device of theinvention in which mixing chambers are present where the lines(representing microchannels) intersect.

FIG. 11 illustrates another embodiment of a microfluidic device of theinvention in which mixing chambers are present where the lines(representing microchannels) intersect.

FIG. 12 illustrates another embodiment of the microfluidic device of theinvention configured to provide multiple aliquots of the same pairwisecombinatorial mixture of first and second liquids.

FIG. 13 illustrates another embodiment of a microfluidic device of theinvention similar to the embodiment of FIG. 10 and in which the wavyline represents the fluid path of a third reagent which is common to allmixing chambers.

FIG. 14 illustrates another embodiment of a microfluidic device of theinvention configured for metering and combinatorial four liquid sampleswith six liquid reagents. In this embodiment, the reservoirs andmetering chambers for the four liquid samples are disposed in a firstplane of the device and the reservoirs and metering chambers for the sixliquid reagents are disposed in a second plane of the device, and themixing chamber (illustrated in FIG. 5) spans the first and secondplanes.

FIG. 15: Where the loading chambers are radially inward of the geometricbarrier during the first centrifugal orientation it is critical that themicrofluidic resistance be higher between the loading chambers and thefirst mixing chambers than further downstream in order to throttle theflow, and prevent overflow of the geometric barriers.

FIG. 16 illustrates an embodiment of the microfluidic chip of theinvention comprising a microtiter plate and an attachment for the plate

FIG. 17: An illustration of how the microfluidic chip of FIG. 16 mayoperate using a standard bucket-centrifuge. By orienting the flatmicrotitre plate at a slight angle in a standard centrifuge acentrifugal force component along the disc. Reversing the plate willreverse the direction of this force as part of the mixing step.Alternatively an automatic system can change the tilt of the plate inthe centrifuge.

FIG. 18: A non-return valve or non-return structure integrated into themixing structure can allow additional reagents to be added subsequent tothe first combinatorial mixing step. It can also enable thecombinatorial mixing to occur in multiple steps. The figure above showsa non-return structure where, following the second rotation, the mixedreagents are trapped in a recessed pocket where the volume will remainat the orientations used for centrifugation.

FIG. 19 illustrates a further embodiment of the device configured forpairwise combinatorial mixing of six first liquids with four secondliquids and having combined metering/mixing chambers as shown in Figure.5. FIG. 19A shows a first layer of the device and the first conduitsproviding fluidic connection from the six central reservoirs to 24metering chambers. FIG. 19B shows a second layer of the device and thesecond conduits providing fluidic connection from the four outermostreservoirs to 24 metering chambers.

FIG. 20: Diamond Designs. (1.) Diamond-1 with circular mixing chambersand capture chambers on both the top and underside of the chip. (2.)Diamond-2 has arc shaped mixing chambers with all the capture chamberson the top of chip. (3.) Diamond-3 has mixing chambers above the loadingchambers and also has all capture chambers on top of chip.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents, patent applications and other referencesmentioned herein are hereby incorporated by reference in theirentireties for all purposes as if each individual publication, patent orpatent application were specifically and individually indicated to beincorporated by reference and the content thereof recited in full.

Definitions and General Preferences

Where used herein and unless specifically indicated otherwise, thefollowing terms are intended to have the following meanings in additionto any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular isto be read to include the plural and vice versa. The term “a” or “an”used in relation to an entity is to be read to refer to one or more ofthat entity. As such, the terms “a” (or “an”), “one or more,” and “atleast one” are used interchangeably herein.

As used herein, the term “comprise,” or variations thereof such as“comprises” or “comprising,” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein the term “comprising” is inclusive oropen-ended and does not exclude additional, unrecited integers ormethod/process steps.

As used herein, the term “disease” is used to define any abnormalcondition that impairs physiological function and is associated withspecific symptoms. The term is used broadly to encompass any disorder,illness, abnormality, pathology, sickness, condition or syndrome inwhich physiological function is impaired irrespective of the nature ofthe aetiology (or indeed whether the aetiological basis for the diseaseis established). It therefore encompasses conditions arising frominfection, trauma, injury, surgery, radiological ablation, poisoning ornutritional deficiencies.

As used herein, the term “fluidic device” means a device having aplurality of different chambers for containing fluids and fluidicchannels providing fluid communication between the chambers. The fluidicdevice may be a microfluidic device. The fluidic device is typically aplanar device. Examples of planar devices include disks, chips andmicrotitre plates. The fluidic device may comprise a plurality oflayers, for example 2, 3, 4, 5, 7, 8, 9 or 10. The fluidic device maycomprise reservoirs for fluids. The reservoirs may comprise aperturesfor filling of the reservoirs with fluid. At least one of the fluidicchannels may include a valving mechanism operable to open and close thefluidic channel. Various types of valving mechanisms may be employed,examples of which are described in the literature referenced herein. Theterm also includes microtitre plates having a plurality and generally amultiplicity of wells, having an associated structure (for example alid) which provides fluidic channels configured to connect the wells ofthe plate to provide a device of the invention. This embodiment of theinvention is described in more detail below. The fluidic device of theinvention may be configured for rotary motion on a rotary device. Whenthe device is a disk, it may have a central aperture (or otherconnection means) for mounting on a turntable. When the device is achip, it may be configured for mounting on a planetary rotation device,typically in at least two different configurations. The fluidic devicemay be performed for any method of procedure requiring combinatorialmixing of liquids including PCR amplification of nucleic acids (forexample thermal or isothermal amplification), arraying of nucleic acids,assaying of chemical or drug libraries (for example to detectinteractions between drugs and ligands), combinatorial chemistry,screening libraries of chemical or biological materials, immunochemicalreactions and the like. In one embodiment, the device is configured forgenotyping samples, especially PCR-based genotyping. In one embodiment,the device is configured for nucleic acid screening for pharmacogenomicor chemotherapeutic screening, for example. In one embodiment, thedevice is configured for nucleic acid microarray hybridisation. In oneembodiment, the device is configured for screening or monitoring ofenvironmental samples. In one embodiment, the device is configured forLAMP amplification of nucleic acid.

As used herein, the term “microfluidic device” means a device comprisingat least one microfluidic channel typically having a diameter of lessthan 1000 microns. The term includes devices configured to performcontinuous flow microfluidics, droplet based microfluidics, digitalmicrofluidics, and for application in nucleic acid arrays andimmuno-assays for clinical and research applications. Typically, thedevice comprises a plurality of microfluidic channels, one or morereservoirs for liquids, and one or more metering and mixing chambers. Inone embodiment, the microfluidic device is a passive device, in whichfluid transport on the chip is effected by means of an external force(for example rotary drives applying centrifugal forces). In oneembodiment, the device may comprise active micro-components such asmicropumps. In one embodiment, the microfluidics device may beconfigured to provide for both active and passive transport of fluidswithin the device. Microfluidics devices are well known in theliterature, and are described in WO2012/164086, U.S. Pat. No. 6,719,682,US2009/166562, WO2006/044841, and US2006/078462. In one embodiment, themicrofluidic device is configured to perform a polymerase chain reaction(PCR). In one embodiment, the microfluidic device is configured toperform an enzyme linked immunosorbent assay (ELISA).

As used herein, the term “combinatorial mixing” refers to mixing eachfirst sample with each second sample. Thus, where the device isconfigured to combinatorially mix three first liquids A, B and C withthree second liquids 1, 2 and 3, combinatorial mixing provides 9pairwise combinations, namely A1, A2, A3, B1, B2, B3, C1, C2 and C3.

As used herein, the term “metering” or “aliquoting” as applied to aliquid sample refers to generating aliquots of the liquid sample. In oneembodiment, a liquid sample is divided into aliquots of equal volume. Inone embodiment, a liquid sample is divided into aliquots of unequalvolume. In one embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10aliquots of a liquid sample are provided. As used herein, the term“metering” refers to providing aliquots of defined volume.

As used herein, the term “spatially separated across the Y-axis of thedevice” as applied to the conduits should be understood to mean that atleast a part of some of the conduits are provided on one plane of thedevice and at least part of other conduits are provided on a secondplane of the device, allowing the conduits to cross-over withoutintersecting by means of Y-axis spatial separation. In one embodiment,some of the conduits may be provided on one plane of the device andother conduits are provided on a second plane of the device. Thus, forexample, the first conduits may be provided on one plane of the deviceand the second conduits may be provided in second plane of the device.This latter embodiment is useful when combined aliquoting/mixingchambers are employed, where a first aliquoting chamber is disposed inone plane of the device and a second aliquoting chamber is disposed in asecond plane of the device (i.e. FIG. 4). In another example, some ofthe third conduits may be provided on a first plane of the device andsome of the third conduits may be provided on a second plane of thedevice. This embodiment is useful where the aliquoting step does notrequire crossing of conduits (i.e. FIG. 9). In another embodiment, atleast some of the conduits may have at least one part in one plane ofthe device and at least a second part in a second plane of the device,and at least one orthogonal component providing fluidic connectionbetween the at least first and second parts. In another embodiment, atleast one of the conduits comprises a distal and proximal part in oneplane of the device, and an intermediate part in a second plane of thedevice, and first and second orthogonal components providing fluidicconnection between the distal and proximal, and intermediate, parts ofthe conduit. In one embodiment, at least one of the conduits comprisesan orthogonal component providing fluidic connection between the conduitand a fluidic chamber (for example a reservoir, aliquoting or meteringchamber, or mixing chamber). In one embodiment, one or more of thereservoirs, aliquoting, or mixing chambers may extend between differentplanes of the device.

As used herein, the term “orthogonal” as applied to a conduit or part ofa conduit refers to a conduit or part thereof that extends out of theplane of the device, and in one embodiment extends generallyperpendicular to a plane of the device. Orthogonal conduits, ororthogonal parts of conduits, are sometimes referred to in the art as“vertical vias” as they extend in the vertical (Y-axis) direction offluidic chips/devices.

As used herein, the term “liquid sample” refers to any liquid sample,including samples of biological material, chemical samples,pharmaceutical samples, chemical reagents, environmental samples.Biological reagents can include cells, cellular extracts or fractions,nucleic acids (i.e. DNA or RNA based nucleic acids), proteins,carbohydrates, antibodies, lipids, metabolites and the like.Pharmaceutical samples can include drugs, libraries of drugs, drugmetabolites, drug candidates, libraries of chemicals obtained bycombinatorial chemistry. Chemical reagents can include reagents involvedin diagnostic assays, for example antigens, antibodies, diagnosticreagents, and chemicals involved in nucleic acid amplification includingpolymerase enzymes and nucleotides. In one embodiment, the liquidsamples include nucleic acid amplification (i.e. PCR) reagents, forexample nucleic acid polymerase enzymes, nucleotides, primers, andprobes. “First liquid sample(s)” refers a set of liquid samples,generally of the same type (i.e. different biological samples) that isto be combinatorially mixed with the second liquid samples. “Secondliquid sample(s)” refers a set of liquid samples, generally of the sametype (i.e. different diagnostic reagents) that is to be combinatoriallymixed with the first liquid samples. Examples of liquid samples includenucleic acids, primers, fluorescent probes.

As used herein, the term “reservoir” means a chamber configured to holda reservoir of the first or second liquid samples. Generally, each ofthe first and second liquid samples has a dedicated reservoir. Eachreservoir has an outlet fluidically connected to a metering chamber(where the metering chamber are provided in series), or a plurality ofmetering chambers (where the metering chambers are provided inparallel). In one embodiment, first reservoirs and second reservoirs areprovided in different planes of the device. In one embodiment, firstand/or second reservoirs extend through different planes of the device.

As used here, the term “M” is a whole number that refers to the numberof first liquid samples to be combinatorially mixed with the secondliquid samples. Typically, M is at least 2, and ideally at least 3, 4,5, 6, 7, 8, 9, or 10. As used here, the term “N” is a whole number thatrefers to the number of first liquid samples to be combinatorially mixedwith the second liquid samples. Typically, M is at least 2, and ideallyat least 3, 4, 5, 6, 7, 8, 9, or 10.

As used herein, the term “aliquot chamber” refers to a chamberconfigured to hold an aliquot of a first or second liquid sample, thatis fluidically connected (directly or indirectly) to a (typically onlyone) reservoir and fluidically connected to a (typically only one)mixing chamber. In one embodiment, the aliquoting chamber is configuredfor metering a liquid sample (i.e. “metering chamber”). In oneembodiment, the metering means comprises an overflow arrangement. In oneembodiment, one or more of the aliquoting/metering chambers are providedin one plane of the device and one or more aliquoting/metering chamberare provided in a second plane of the device.

As used herein, the term “conduit” refers to a passageway configured toprovide fluid communication between one location (i.e. a reservoir) anda second location (i.e. a metering chamber). In one embodiment, at leastone or more of the conduits are microfluidic conduits. In oneembodiment, all of the conduits are microfluidic conduits. Typically,first conduits are provided between the first reservoirs and firstaliquoting/metering chambers (or between adjacent aliquoting/meteringchambers when they are aligned in series) and typically second conduitsare provided between the second reservoirs and secondaliquoting/metering chambers (or between adjacent aliquoting/meteringchambers when they are aligned in series). The first and second conduitsmay be disposed in different planes of the device to allow cross-over ofthe conduits). Typically, third conduits are provided betweenaliquoting/metering chambers and mixing chambers. Some of the thirdconduits may be disposed in one plane of the device and other thirdconduits may be disposed in a second plane of the device.

As used herein, the term “mixing chamber” refers to a chamber configuredto hold an aliquot of a first liquid sample and an aliquot of a secondliquid sample and combine the samples when the chip is exposed to thesecond directional force field. In one embodiment, the mixing chambersare remote from the aliquoting/metering chambers and connected by meansof conduits, especially microfluidic conduits. In one embodiment, one ormore of the mixing chambers extends between at least two planes of thedevice. In one embodiment, M*N mixing chambers are provided. In oneembodiment, the or each mixing chamber is configured to receive analiquot of a first liquid sample and an aliquot of a second liquidsample. Thus, N*M combinations of aliquots is provided.

As used herein, the term “trap” (or “non-return valve” or “non-returnstructure”) as applied to a mixing chamber refers to a configuration ofmixing chamber that prevents the mixed aliquots being returned to themetering chambers when the device is exposed to the first directionalforce field. An embodiment of a mixing chamber with a trap is shown inFIG. 16, although different embodiment will be apparent to a personskilled in the art.

As used herein, the term “first orientation relative to the device” asapplied to the first and second conduits refers to a configuration ofthe conduits that permits fluid flow in the conduits from the reservoirto the metering chambers when the device is exposed to the firstdirectional force field. Likewise, the term “second orientation relativeto the device” as applied to the third conduits refers to aconfiguration of the conduits that permits fluid flow in the conduitsfrom the metering chambers to the mixing chambers when the device isexposed to the second directional force field.

As used herein, the term “in parallel” as applied to a reservoir meansthat reservoir is directly fluidically connected to each of the N firstaliquoting/metering chambers.

As used herein, the term “in series” as applied to a reservoir meansthat an aliquoting/metering chamber A is fluidically connected to thereservoir, metering chamber B is fluidically connected toaliquoting/metering chamber A, aliquoting/metering chamber C isfluidically connected to aliquoting/metering chamber B, etc.), providinga cascading flow of liquid from the reservoir to the metering chambers.In one embodiment, the metering chambers are fluidically connected bymeans of an overflow mechanism which serves to meter the liquid in themetering chamber. Typically, the overflow mechanism is provided by anoutlet in the metering chamber which defines the volume of liquidretained in the metering chamber. This is illustrated in FIG. 5C. In oneembodiment, the final metering chamber in the series has an overflowfluidically connected to a waste chamber.

As used herein, the term “single chamber” or “metering/mixing chamber”(or “aliquoting/mixing chamber”) refers to an embodiment of the devicein which at least one mixing chamber (and associated aliquoting/meteringchambers) is provided a single chamber having two aliquoting/meteringregions and one mixing region. Metering/mixing chambers are illustratedin FIGS. 4, 5, 12 and 13. The device of FIG. 4 has an arched-typestructure (for example a U-shape or V-shape), with two feet comprisingthe metering regions, and an apex comprising the mixing region. In oneembodiment, the metering regions and mixing region are spaced-apart. Inone embodiment, the metering/mixing chamber is disposed in one plane ofthe device. In another embodiment (see FIG. 12 as an example), onemetering chamber is disposed in one plane of the device and anothermetering chamber is disposed in another plane of the device, and themixing chamber typically extends between the two planes. Thisembodiment, allows the fluidic architecture feeding a first set ofmetering chambers (i.e. N metering chamber) be disposed in one plane ofthe device and the fluidic architecture feeding the second set ofmetering chamber (i.e. M metering chambers) be disposed in a secondplane of the device, which in turn allows the conduits cross each other.

As used herein, the term “directional force field” refers to a forcethat the device is subjected to that, in use, causes one or more of thefluids in the device to move. Examples include force fields generated mymovement of the device (i.e. centrifugal force fields, inertia forcefield) or force fields that do not require movement of the device (i.e.gravity). Examples include centrifugal force fields, gravity, a forcefield generated by pressure changes (or pressure gradients) in theliquids, a force field generated by temperature changes (or temperaturegradients) in the liquids, mechanical pump generated force fields. In apreferred embodiment, the directional force field is selected from acentrifugal or gravity based force field. In one embodiment, the firstdirectional force field is one type of force field (i.e. centrifugal)and the second directional force field is a second type of force field(i.e. gravity). In one embodiment, both the first and second force fieldare of the same type (i.e. gravitational force fields). In oneembodiment, the first and second directional force fields are the sameforce field, where re-orientation of the device with respect to theforce field causes the device to be exposed to different directionalforce fields. In one embodiment, the directional force field is acentrifugal force field.

The device of the invention, or the layers of the device, may be formedfrom a suitable polymeric material, for example a thermoplastic resin.Examples of thermoplastic resins include cyclic olefin copolymers,polymethylmethacrylate, polycarbonate, polystyrene, polyoxymethylene,perfluoralkoxy, polyvinylchloride, polypropylene, polyethyleneterephthalate, polyetheretherketone, polyamide, polysulphone, andpolyvinylidine chloride. The device may be a planar device, of anyshape. The device may comprise a layered structure, in which the layersare optionally adhered together with adhesive, for example a pressuresensitive adhesive. In one embodiment, at least some of the layers maybe formed from polymethylmethacrylate (PMMA). The structures formed onthe device (i.e. the conduits, reservoirs, etc.) may be formed in anymanner—exemplary methods include use of a knife-cutting machine or CO²laser cutting. In one embodiment, each layer of the device has athickness of 0.1 to 10 mm, preferably 0.1 to 5 mm, more preferably 0.5to 5 mm.

Exemplification

The invention will now be described with reference to specific Examples.These are merely exemplary and for illustrative purposes only: they arenot intended to be limiting in any way to the scope of the monopolyclaimed or to the invention described. These examples constitute thebest mode currently contemplated for practicing the invention.

The centrifugal microfluidic chip claimed uses the principle ofdirecting liquid about the cartridge based on a re-orientation of thedisc in a directed force field. While (terrestrial) gravity and similardirectional fields may be employed, we describe here a mechanism basedof the rotationally induced centrifugal field for the sake of clarity.From the state-of-the-art, it is evident where the centrifugal field maybe replaced, either entirely of for parts of the proposed methods, byother density dependent, unidirectional fields.

This orientation of the field can be by means of rotation (FIG. 1), e.g.by 180 to achieve a complete reversal of the initial force field (FIG.2). This re-orientation can, for example, be achieved by means of asecondary spindle motor, by means of changing the centrifugal force toaction a spring or to open a latch (FIG. 3). Similarly, this change inorientation can take place when the cartridge is in motion or by firststopping the rotor, re-orienting the cartridge and resuming thespinning. When the centrifuge is stopped, the chip may be devised byprinciples know to the expert in the field so forces induced by surfaceor interfacial tension surface which tend to dominate towards the smalllength scales in microfluidics retain the liquid elements in theircollection pockets until another body force, e.g. induced by asubsequent centrifuge step, is applied. In another teaching thecartridge can be manually repositioned before the second centrifugationstep (i.e. FIG. 7)

Referring to FIGS. 4 and 5, one embodiment of a microfluidic device(chip) of the invention is described in detail. The chip is shown in anexpanded view illustrating the different layers of the device and theseparation of the first and second conduits into different layers of thedevice (Y-axis dimensional bridging). The layers of FIGS. 4A and 4G arefront and rear cover layers, respectively, the front cover havingapertures 2 for filling the first reservoirs 3A and second reservoirs3B. The layer of FIG. 4B contains the first conduits 4A providingfluidic connection between the first reservoirs 3A and combinedmetering/mixing chambers 5. The layer of FIG. 4F contains the secondconduits 4B providing fluidic connection between the second reservoirs3B and metering/mixing chambers 5. Each metering/mixing chamber 5 has afirst metering region 6A in one layer (FIG. 4B) in fluid communicationwith the first conduits 4A, a second metering region 6B in a secondlayer (FIG. 4F) in fluid communication with the second conduits 4B, anda mixing chamber 7 which spans the layers of FIGS. 4B to 4F. Eachreservoir is connected to a four metering mixing chambers, arranged inseries and connected by means of overflow mechanisms which serve tometer a defined aliquot of liquid into the metering regions of eachmetering/mixing chamber. The last metering/mixing chamber is fluidicallyconnected to an outlet chamber 8. FIGS. 4C to 4E illustrates how thecircular mixing region of the metering/mixing chamber spans the threelayers of FIGS. 4C to 4E, and one metering chamber is disposed in thelayer of FIG. 4C and one is disposed in the later of FIG. 4E, whichensures that the metered aliquots in the metering chambers are keptseparate until a second directional force is applied which forced therespective aliquots towards the mixing region where they combine.

Referring to FIG. 5, a combined metering/mixing chamber of the typeemployed in the microfluidic chip of FIG. 4 is illustrated in moredetail in which parts identified with reference to FIG. 4 are assignedthe same reference numerals. The orthogonal view provided illustrateshow the metering regions 6A and 6B of the metering/mixing chamber 5 areprovided in different planes (layers) of the chip, which allows forY-axis dimensional bridging of the conduits 4A and 4B. This figure alsoillustrates the overflow mechanism that is provided for metering adefined aliquot into each metering chamber and allowing the firstmetering regions and second metering regions to be connected in series.The metering region (capture pocket) is defined by the position of theoutlet (overflow) conduit.

FIG. 6 illustrates an alternative embodiment of a metering/mixingchamber that is similar to that of FIG. 5 but comprises four meteringregions separated along the Y-axis of the chip and a common mixingchamber which spans the Y-axis of the device.

Referring to FIGS. 7 and 8, an alternative embodiment of the inventionis described which is similar in construction to the chip described withreference to FIG. 4. In this embodiment, three first liquids (samples)are combinatorially mixed with three reagents (second liquids). Althoughnot illustrated, the device has a similar layered structure to thedevice of FIG. 4, where the first conduits and second conduits areprovided on different layers of the device, providing Y-axis dimensionalbridging. The chip is shown mounted on the arm of a planetary rotordevice configured to rotate the arm and expose the chip to a centrifugalforce field. FIG. 7A shows the samples and reagents being added to thesix reservoirs. FIG. 7B shows the metering of the samples and reagentsinto the metering regions of the metering/mixing chambers, due torotation of the arm and resultant centrifugal force being exerted on thechip. In FIG. 7C, the chip has been re-positioned on the arm (rotatedabout 180 degrees) so that the centrifugal force exerted, relative tothe chip, is reversed, and this causes aliquots of sample and reagent tobe forced into the mixing regions of the metering/mixing chambers.

The combined metering/mixing chambers of this device are different tothose described previously, and are described in detail in FIG. 8. Thecombined metering/mixing chamber of this embodiment, is an arch-shapeddevice having legs which provide metering regions (capture pockets) andan apex which provides a mixing region. Application of the firstdirectional force field (FIG. 7B) forces sample and reagent into therespective metering regions, and application of the second directionalforce field (FIG. 7C) forces sample and reagents from the meteringregions into the mixing regions.

In the foregoing embodiments, combinatorial mixing can be achieved usinga centrifugally driven microfluidic chip. In one exemplaryconfiguration, a chip features a plurality of M inlets for a reagent (P)of one type, and a number of inlets (N) for a reagent of a second type(S). Centrifuging the chip will pump P and S through the chip such thata defined volume of P and S is metered in each captured chamber. Theindividual defined volumes of P and S are prevented from mixing via ageometric barrier. By their length or cross section, the hydrodynamicresistances of the microchannels between the loading chambers and themixing chambers are designed to prevent the reagents overflowing thegeometric barriers during this metering step (FIGS. 4 and 7). Followingthe first centrifugal aliquoting step, re-orientating the chip in thecentrifugal field drives the liquid into a section of each mixing chipwhere they can mix.

Referring to FIG. 9, there is illustrated an alternative embodiment ofthe microfluidic device of the invention. In this embodiment, the deviceis configured to combinatorially mix one sample (first liquid) with tworeagents (second liquids). In this embodiment, separate metering andmixing chambers are provided, and the third conduit providing fluidicconnection between the metering and mixing chambers extends generallyradially inwardly with respect to the rotor when the device is in thefirst orientation with respect to the rotor, ensuring that liquid doesnot overflow the metering chambers and flow into the mixing chamberswhen the first directional (centrifugal) force is applied. In thisembodiment, one first liquid is metered from a first reservoir (loadingchamber) into two adjacent metering chamber (capture pockets), and twosecond liquids are metered into metering chambers (capture pockets) bythe application of centrifugal force in a first direction. The positionof the device with respect to the centrifugal field is then reversed,which causes the first and second liquids in the capture pockets to moveradially inward towards the mixing chambers, where the liquids combine.

The architecture of the chip to enable mixing of M aliquots of onereagent with N aliquots of another can take a number of configurations.For example, one set of linking channels might be predominately verticaland the other predominantly horizontal (FIG. 10). Similarly, both mightbe horizontal or both might be in a diagonal configuration (FIG. 11). Inone embodiment, the device is configured to provide a plurality ofaliquots of each combinatorial mixture of first and second liquids (FIG.12).

In an alternative configuration, additional reagents common to allcombinatorial mixing chambers might be added through a dedicated channel(FIG. 13). For example, the ‘M’ loading chambers might containbiological samples containing DNA, the ‘N’ loading chamber might containprimers for PCR or an isothermal DNA amplification technique such asLAMP. The ‘common’ loading chamber channel might deliver common reagentssuch as fluorescence markers and DNA amplification enzymes (such as TaqPolymerase).

FIG. 14 illustrates an alternative embodiment of the device of theinvention which is similar to the device illustrated in FIG. 4.

In cases where the loading chambers are placed radially inwards of thegeometric barriers, the use of inbound microchannels with higher fluidicresistance is critical as this will throttle the flow through the chipand thus prevent overflow of the geometrical barriers which permit thesamples to be metered without mixing during the initial centrifugation(FIG. 15

In one embodiment, the microfluidic device of the invention is formedfrom a conventional microtiter plate and an attachment for themicrotiter plate that closes the wells and provides microfluidicconnections between the wells of the plate. An embodiment is illustratedin FIG. 16 showing the wells 11 of the microtiter plate and anattachment 12 that is configured to attach to the plate and close thewells 11. The attachment 12 has a central section 13 having downwardlydepending spouts 14, a first end section 15 having a plurality ofupwardly depending wells, namely eight first reservoirs 16A and twelvesecond reservoirs 16B, and a second end section 17 having eight wasteliquid chambers 18A and twelve waste liquid chambers 18B. The centralsection 13 contains metering chambers and first and second microfluidicconduits (not shown) configured to meter the first and second liquidsinto aliquots. Each metering chamber is fluidically connected to one ofthe downwardly depending spouts 14. The spouts are configured so thatwhen the attachment 11 is attached to the microtiter plate, two spouts14 will project into each well 11 of the microtiter plate to provide thetwo aliquots to be mixed, where the wells 11 act as the mixing chambers.The attachment is configured such that when it is exposed to a firstdirectional force field, the liquids are forced from the reservoirs intothe metering chambers, and when the device is exposed to a seconddirectional force field, the liquids are forced from the meteringchambers into the mixing chambers.

A method of using the device of FIG. 16 is described with reference toFIG. 17. In this embodiment, the device is assembled and the reservoirsare loaded with liquids, before the device is attached to a swingingbucket rotor centrifuge. The device is offset (i.e. 45 degrees) so thata component of the centrifugal force acts laterally. The device iscentrifuged in this position, forcing liquids from the reservoirs intothe metering chambers. The rotor is then stopped and the device ismanually re-positioned as shown, and a second centrifuge step is carriedout which forces liquid from the metering chambers into the wells of themicrotiter plate through the spouts, where aliquots of the liquids mix.

In another embodiment, illustrated in FIG. 18, the integration of anon-return geometry or non-return valve into the mixing chambers canenable the liquids be added to the chip in multiple steps or theaddition of reagents in stages. For example, in the PCR case, the DNApolymerase might be loaded in a first step using one of two sets ofloading inlets. In the second step, the primers can be loaded in usingthe same of loading inlets while the samples can be loaded through thesecond set of loading inlets. Thus the three components required can beadded sequentially.

FIG. 19 illustrates a further embodiment of the device configured forpairwise combinatorial mixing of six first liquids with four secondliquids and having combined metering/mixing chambers as shown in Figure.5. FIG. 19A shows a first layer of the device and the first conduitsproviding fluidic connection from the six central reservoirs to 24metering chambers. FIG. 19B shows a second layer of the device and thesecond conduits providing fluidic connection from the four outermostreservoirs to 24 metering chambers.

FIG. 20 illustrates devices of the invention having “diamond array”configurations of reservoirs and metering/mixing chambers (FIGS. 20.1and 20.2) and a diamond array of metering chambers (FIG. 20.3). Theembodiment of FIG. 20.1 has combined metering/mixing chambers as shownin FIG. 5. The embodiment of FIG. 20.2 has combined metering/mixingchambers as shown in FIG. 8.

The fluidic device may be employed for any method or procedure requiringcombinatorial mixing of liquids including PCR amplification of nucleicacids (for example thermal or isothermal amplification), arraying ofnucleic acids, assaying of chemical or drug libraries (for example todetect interactions between drugs and ligands), combinatorial chemistry,screening libraries of chemical or biological materials, immunochemicalreactions, PCR-based genotyping, nucleic acid screening forpharmacogenomic or chemotherapeutic screening, nucleic acid microarrayhybridisation, screening or monitoring of environmental or forensicsamples, or LAMP amplification of nucleic acids.

EQUIVALENTS

The foregoing description details presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare intended to be encompassed within the claims appended hereto.

REFERENCES

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The invention claimed is:
 1. A fluidic device for aliquoting andpairwise combinatorial mixing of at least two first liquid samples withat least two second liquid samples, the microfluidic device comprising:a first reservoir for each of the at least two first liquid samples anda second reservoir for each of the at least two second liquid samples;at least two first aliquot chambers fluidically connected to each offirst reservoirs by first microfluidic conduits having a firstorientation relative to the device; at least two second aliquot chambersfluidically connected to each of the second reservoirs by secondmicrofluidic conduits having a first orientation relative to the device;at least four mixing chambers, in which each mixing chamber isfluidically connected to at least one of the first aliquot chambers andat least one of the second aliquot chambers by third microfluidicconduits having a second orientation relative to the device, wherein thefirst and second microfluidic conduits are configured such thatapplication of a first rotor-driven unidirectional centrifugal forcefield effects liquid flow in the first orientation to generate aliquotsof the at least two first liquid samples in the first aliquot chambersand the at least two second liquid samples in the second aliquotchambers, wherein the third microfluidic conduits are configured suchthat application of a second rotor-driven unidirectional centrifugalforce field effects liquid flow in the second orientation tocombinatorially combine aliquots of the at least two first liquidsamples with aliquots of the at least two second liquid samples in apairwise manner, wherein the third microfluidic conduits are configuredto extend against the direction of the first rotor-driven unidirectionalcentrifugal force field to prevent movement of liquid from the firstaliquot chambers and second aliquot chambers to the mixing chambersduring application of the first rotor-driven unidirectional centrifugalforce field, wherein at least some of the conduits are spatiallyseparated across the Y-axis of the device.
 2. The fluidic deviceaccording to claim 1 in which the fluidic device is a microfluidicdevice.
 3. The fluidic device according to claim 1 in which one or moreof the microfluidic conduits has at least one part in one plane of thedevice and at least a second part in a second plane of the device, andat least one orthogonal component providing fluidic connection betweenthe at least first and second parts.
 4. The fluidic device according toclaim 1 in which some of the microfluidic conduits are provided in oneplane of the device and other microfluidic conduits are provided in asecond plane of the device.
 5. A fluidic device according to claim 1 inwhich the first microfluidic conduits are disposed in a first plane ofthe device and the second microfluidic conduits are disposed in a secondplane of the device.
 6. The fluidic device according to claim 1 in whichat least one of the first reservoirs is fluidically connected to the atleast two first aliquot chambers in series, wherein the first aliquotchambers are fluidically connected by means of an overflow mechanismconfigured to meter a defined aliquot volume into the aliquot chambers.7. The microfluidic device according to claim 1 in which at least one ofthe first reservoirs is fluidically connected to the at least two firstaliquot chambers in series, wherein the first aliquot chambers arefluidically connected by means of an overflow mechanism configured tometer a defined aliquot volume into the aliquot chambers, and in which afinal aliquot chamber in the series has an overflow fluidicallyconnected to a waste chamber.
 8. The fluidic device according to claim 1in which at least one of the mixing chambers and fluidically connectedfirst and second aliquot chambers are provided by a single chamber(aliquot/mixing chamber) having aliquot regions and a mixing regiondisposed such that application of the second directional force fieldeffects liquid flow from the aliquot regions to the mixing region. 9.The fluidic device according to claim 1 in which at least one of themixing chambers and fluidically connected first and second aliquotchambers are provided by a single chamber (aliquot/mixing chamber)having aliquot regions and a mixing region disposed such thatapplication of the second directional force field effects liquid flowfrom the aliquot regions to the mixing region, and in which the aliquotregions and mixing region are disposed on opposite ends of the singlechamber.
 10. The fluidic device according to claim 1 in which at leastone of the mixing chambers comprises a trap structure configured toprevent liquid in the mixing chamber returning to the associated aliquotchambers upon application of the first directional force field.
 11. Thefluidic device according to claim 1, in which at least one of the mixingchambers comprises a trap structure configured to prevent liquid in themixing chamber returning to the associated aliquot chambers uponapplication of the first directional force field, and including a liquidcollection chamber fluidically connected the trap structures of themixing chamber by collection conduits, wherein the collection conduitsare configured to force the liquid in the mixing chambers to thecollection chamber upon application of a directional force field. 12.The fluidic device according to claim 1 including a third reservoir fora third liquid sample, and a conduit configured to deliver an aliquot ofthe third liquid sample to one or more of the mixing chambers.
 13. Thefluidic device according to claim 1 in which the first and secondmicrofluidic conduits are configured to have a higher fluidic resistancethan the third microfluidic conduits.
 14. The fluidic device accordingto claim 1 in which the third microfluidic conduits have a radiallyinward configuration.
 15. The fluidic device according to claim 1, inwhich the combined aliquot/mixing chambers are arrayed in a diamondconfiguration.
 16. The fluidic device according to claim 1 in the formof a planar chip.
 17. The fluidic device according to claim 1 comprisinga microtitre plate having a multiplicity of wells, and an attachment forthe plate comprising the reservoirs, aliquot chambers, and conduits,wherein the wells of the plate are the mixing chambers.
 18. The fluidicdevice according to claim 17 comprising a microtitre plate having amultiplicity of wells, and an attachment for the plate comprising thereservoirs, aliquot chambers, and conduits, wherein the wells of theplate are the mixing chambers, and in which the attachment comprises afirst region comprising the reservoirs, a second region configured toattach to a top of the microtiter plate to close some or all of thewells of the plate, wherein an underside of the second region comprisesa plurality of spouts configured to project into wells of the plate whenthe device is assembled.
 19. The microfluidic device according to claim1 in which the aliquot chambers are metering chamber and in which thecombined aliquot/mixing chambers are combined metering/mixing chambers.20. A microfluidic fluidic device for aliquoting and pairwisecombinatorial mixing of at least three first liquid samples with atleast three second liquid samples, the microfluidic device comprising:six reservoirs comprising a first reservoir for each of the three firstliquid samples and a second reservoir for each of the three secondliquid samples; eighteen aliquot chambers comprising: three firstaliquot chambers fluidically connected to each of the first reservoirsby first microfluidic conduits having a first orientation relative tothe device; three second aliquot chambers fluidically connected to eachof the second reservoirs by second microfluidic conduits having a firstorientation relative to the device; nine mixing chambers, in which eachmixing chamber is fluidically connected to at least one of the firstaliquot chambers and at least one of the second aliquot chambers bythird microfluidic conduits having a second orientation relative to thedevice, wherein the first and second microfluidic conduits areconfigured such that application of a first rotor-driven unidirectionalcentrifugal force field effects liquid flow in the first orientation togenerate three aliquots of each of the three first liquid samples in thefirst aliquot chambers and three aliquots of each of the three secondliquid samples in the second aliquot chambers, wherein the thirdmicrofluidic conduits are configured such that application of a secondrotor-driven unidirectional centrifugal force field effects liquid flowin the second orientation to combinatorially combine aliquots of thethree-first liquid samples with aliquots of the three second liquidsamples in a pairwise manner, wherein the third microfluidic conduitsare configured to extend against the direction of the first rotor-drivenunidirectional centrifugal force field to prevent movement of liquidfrom the first aliquot chambers and second aliquot chambers to themixing chambers during application of the first rotor-drivenunidirectional centrifugal force field, wherein at least some of theconduits are spatially separated across the Y-axis of the device.
 21. Afluidic device for aliquoting and pairwise combinatorial mixing of atleast two first liquid samples with at least two second liquid samples,the microfluidic device comprising: a first reservoir for each of the atleast two first liquid samples and a second reservoir for each of the atleast two second liquid samples; at least two first aliquot chambersfluidically connected to each of first reservoirs by first microfluidicconduits having a first orientation relative to the device; at least twosecond aliquot chambers fluidically connected to each of the secondreservoirs by second microfluidic conduits having a first orientationrelative to the device; at least four mixing chambers, in which eachmixing chamber is fluidically connected to at least one of the firstaliquot chambers and at least one of the second aliquot chambers bythird microfluidic conduits having a second orientation relative to thedevice, wherein the first and second microfluidic conduits areconfigured such that application of a first rotor-driven unidirectionalcentrifugal force field effects liquid flow in the first orientation togenerate aliquots of the at least two first liquid samples in the firstaliquot chambers and the at least two second liquid samples in thesecond aliquot chambers, wherein the third microfluidic conduits areconfigured such that application of a second rotor-driven unidirectionalcentrifugal force field effects liquid flow in the second orientation tocombinatorially combine aliquots of the at least two first liquidsamples with aliquots of the at least two second liquid samples in apairwise manner, wherein at least some of the conduits are spatiallyseparated across the Y-axis of the device, and wherein the first andsecond microfluidic conduits are configured to have a higher fluidicresistance than the third microfluidic conduits.
 22. A fluidic devicefor aliquoting and pairwise combinatorial mixing of at least two firstliquid samples with at least two second liquid samples, the microfluidicdevice comprising: a first reservoir for each of the at least two firstliquid samples and a second reservoir for each of the at least twosecond liquid samples; at least two first aliquot chambers fluidicallyconnected to each of first reservoirs by first microfluidic conduitshaving a first orientation relative to the device; at least two secondaliquot chambers fluidically connected to each of the second reservoirsby second microfluidic conduits having a first orientation relative tothe device; at least four mixing chambers, in which each mixing chamberis fluidically connected to at least one of the first aliquot chambersand at least one of the second aliquot chambers by third microfluidicconduits having a second orientation relative to the device, wherein thefirst and second microfluidic conduits are configured such thatapplication of a first rotor-driven unidirectional centrifugal forcefield effects liquid flow in the first orientation to generate aliquotsof the at least two first liquid samples in the first aliquot chambersand the at least two second liquid samples in the second aliquotchambers, wherein the third microfluidic conduits are configured suchthat application of a second rotor-driven unidirectional centrifugalforce field effects liquid flow in the second orientation tocombinatorially combine aliquots of the at least two first liquidsamples with aliquots of the at least two second liquid samples in apairwise manner, wherein at least some of the conduits are spatiallyseparated across the Y-axis of the device, and including a thirdreservoir for a third liquid sample, and a conduit configured to deliveran aliquot of the third liquid sample to the at least four mixingchambers.